Voltage activated calcium channels play important roles including neuroexcitation, neurotransmitter and hormone secretion, and regulation of gene transcription through Ca-dependent transcription factors. Their functions depend in part on their cellular localization and their gating properties (characteristics of their opening, inactivation, deactivation, and recovery from inactivation). Five general classes of voltage activated calcium channels have been observed in various neuronal and non-neuronal tissues. The complement of channel subunits and the subcellular localization of the expressed voltage activated calcium channels determine the functional cellular properties.
Diversity of Voltage-gated Ca Channels Fall into Two Major Categories: Low Voltage Activated (LVA) and High Voltage Activated (HVA)
A conserved general structure for all cloned voltage-gated calcium channel alpha subunits (the pore-forming subunit) has been identified. It consists of 4 domains with homology to the domains present in voltage-gated K and Na channels. Each domain contains 6 membrane spanning regions (S1-S6) and a pore region (P) located between S5 and S6. The extracellular loops are generally very short; intracellular loops contain sites that are modulated by phosphorylation and can interact with other effectors. However, there are notable differences in the lengths of the S5-S6 loop of domain I and the intracellular loop between domains I and II among alpha subunits.
Different calcium channels are best distinguished by their pharmacological profiles since their electrophysiological properties differ depending on the cell type or tissue in which they are expressed, presumably because of modulation by cellular proteins, for instance kinases, and also auxiliary calcium channel subunits.
The HVA channel classes are thought to be composed of at least 3 or 4 different subunits: xcex11 (which contains the pore), beta (xcex2) and xcex12xcex4. In skeletal muscle a xcex3 subunit also co-precipitates with the skeletal channel complex. Recently two gamma-like subunits have been cloned from brainxe2x80x94one of which is the gene mutated in the stargazer mutant mouse (Black et al., 1999; Letts et al., 1998). The subunit composition has been proved for only the skeletal L-type (xcex11 xcex12xcex4 xcex2 xcex3) and brain N-type (xcex11 xcex12xcex4 xcex2) channels (Perez-Reyes and Schneider, 1995). These channels generally require large membrane depolarizations for activation (xcx9c30 mV from the resting potential (RP)). Four classes of HVA calcium channels have been identified on the basis of electrophysiological, pharmacological and molecular data. These classes include L-type (encoded by at least 4 genes (including a xcex11 subunits xcex11S (skeletal muscle), xcex11C, xcex11D (neuroendocrine), and xcex11F (retinal)), N-type (xcex11B; (Williams et al., 1992)), P/Q-type (xcex11A) and R-type (encoded by at least the xcex11E gene).
HVA xcex11 families are strongly affected by co-expression of the cytoplasmically localized xcex2 subunit, particularly the expression levels of functional cell surface channels and the electrophysiological response of the channel (ie., kinetics). xcex2 subunits interact with a specific sequence in the I-II intracellular loop to increase the number of functional channels and alter the activation and inactivation properties of the channel complex (Furukawa et al., 1998). There are at least 4xcex2 genes that are alternatively spliced (xcex21a-c; xcex22a-c; xcex23; xcex24;(Perez-Reyes and Schneider, 1995)); the effect of each of these xcex2s on xcex11 function appears to depend on the xcex11 class. Interestingly, mutants in xcex2 (Cchxcex24) produce ataxia and seizures in the lethargic (lh) mouse (Burgess et al., 1997). xcex12xcex4 subunits also modulate xcex11 function and the known gene co-segregates with malignant hyperthermia phenotype in certain families (Iles et al., 1994).
The physiological roles of HVA channels depend on subcellular location of the channel and tissue type. Subcellular location varies among tissues but have been shown to be important in neurotransmitter and hormone release, action potential duration, excitation-contraction coupling in muscle cells, and gene expression (Miller, 1987).
There are at least three genes in the T-type family of LVA calcium channels (xcex11G, xcex11H, and xcex11I) (Perez-Reyes, 1998). Their structure differs from that of the HVA channels in a number of important ways. The I-II intracellular linker is much longer (xcx9c400 amino acids) than that of the known HVA channels. The Domain I S5-P extracellular linker is longer than that of the HVA channels and may be a good target for drug interactions with this channel. xcex2 does not appear to be associated with xcex11 in this class and they lack the canonical sequence that is known to be crucial for beta subunit binding (Lambert et al., 1997; Leuranguer et al., 1998). Anti-sense experiments directed against all known beta""s show a decrease in the expression of HVA calcium channels but not LVA calcium channels in nodose ganglion neurons (Lambert et al., 1997).
Other proteins or cellular environments may be required for robust T-channel expression since xcex11G expressed in oocytes or HEK293 cells produces dramatically different current magnitudes in these two cell types (Perez-Reyes, 1998).
T-type calcium currents have been observed in vivo in many cell types in the peripheral and central nervous systems including thalamus, inferior olive, cerebellar Purkinje cells, lateral habenular cells, dorsal horn neurons, sensory neurons (DRG, nodose), cholinergic forebrain neurons, hippocampal intemeurons, CA1, CA3 dentate gyrus pyramidal cells, basal forebrain neurons, amygdaloid neurons (Talley et al., 1999). T-type channels are prominent in the soma and dendrites of neurons that reveal robust Ca-dependent burst firing behaviors such as the thalamic relay neurons and cerebellar Purkinje cells (Huguenard, 1996).
Physiological Roles and Therapeutic Areas
T-type calcium channels are involved in the generation of low threshold spikes to produce burst firing (Huguenard, 1996). These channels differ from HVA channels in that they have some probability of opening at the resting membrane potential. Because their steady state inactivation curve is shifted toward negative voltages compared to HVA channels (ie., half the channels are not inactivated and are able to be opened by a depolarizing voltage step at voltages more negative than the resting membrane potential (RP)), there is a window current near the RP (ie., a portion of the T-channels are open at RP). Low threshold spikes and rebound burst firing is prominent in neurons from inferior olive, thalamus, hippocampus and neocortex (Huguenard, 1996).
T-type channels promote oscillatory behavior which has important consequences for epilepsy. The ability of a cell to fire low threshold spikes is critical in the genesis of oscillatory behavior and increased burst firing (groups of action potentials separated by about 50-100 ms). T-type calcium channels are thought to play a significant role in absence epilepsy, a type of generalized non-convulsive seizure. The evidence that voltage-gated calcium currents contribute to the epileptogenic discharge, including seizure maintenance and propagation includes 1) a specific enhancement of T-type currents in the reticular thalamic (nRT) neurons which are hypothesized to be involved in the genesis of epileptic seizures in a rat genetic model (GAERS) for absence epilepsy (Tsakiridou et al., 1995); 2) antiepileptics against absence petit mal epilepsy (ethosuximide and dimethadione) have been shown at physiologically relevant doses to partially depress T-type currents in thalamic (ventrobasal complex) neurons (Coulter et al., 1989; Kostyuk et al., 1992); and 3) T-type calcium channels underlie the intrinsic bursting properties of particular neurons that are hypothesized to be involved in epilepsy (nRT, thalamic relay and hippocampal pyramidal cells) (Huguenard, 1996). The rat xcex11G is highly expressed in thalamocortical relay cells (TCs) which are capable of generating prominent Ca2+-dependent low-threshold spikes (Talley et al., 1999).
T-type channels play a critical role in thalamic oscillations and cortical synchrony, and their involvement has been directly implicated in the generation of cortical spike waves that are thought to underlie absence epilepsy and the onset of sleep (McCormick and Bal, 1997). Oscillations of neural networks are critical in normal brain function such during sleep-wave cycles. It is widely recognized that the thalamus is intimately involved in cortical rhythmogenesis. Thalamic neurons most frequently exhibit tonic firing (regularly spaced spontaneous firing) in awake animals, whereas phasic burst firing is typical of slow-wave sleep and may account for the accompanying spindling in the cortical EEG. The shift to burst firing occurs as a result of activation of a low threshold Ca2+ spike which is stimulated by synaptically mediated inhibition (ie., activated upon hyperpolarization of the RP). The reciprocal connections between pyramidal neurons in deeper layers of the neocortex, cortical relay neurons in the thalamus, and their respective inhibitory interneurons are believed to form the elementary pacemaking circuit.
T-type channels contribute to synaptic potentiation at the postsynaptic level since small changes in membrane potential (Vm) (either depolarizations (epsps; excitatory postsynaptic potentials) or hyperpolarizations (ipsps (inhibitory postsynaptic potentials); anode break exhaltation or rebound burst firing) can open T-type calcium channels. At the hyperpolarized Vm during the ipsp more T-type channels become available to open (they have recovered from inactivation) so that upon repolarization to the RP, a larger proportion of T channels are opened and this produces anode break exhaltation, a robust rebound burst firing as the low threshold Ca spike reaches threshold for Na channel activation and action potential generation. A burst of action potentials ride on top of the Ca-dependent depolarization. This phenomenon is particularly prominent in reticular thalamic neurons (Huguenard, 1996).
T-type channels can be involved in transmitter release. In cells where T-channels are located at the presynaptic terminal, they promote neurotransmitter release (Ahnert-Hilger et al., 1996; Arnoult et al., 1997)
T-type channels contribute to spontaneous fluctuations in intracellular Ca concentrations [Ca]i. They are important in pacemaker activity and therefore heart rate in the heart, and in vesicle release from non-excitable cells (Ertel et al., 1997).
T-type calcium currents are expressed differentially in different subpopulations of adult rat dorsal root ganglion (DRG) neurons. T-type currents were present at moderate densities in small diameter Type 1 and 3 cells, the former having TTX-resistant Na currents, long duration action potentials and capsaicin sensitivity (consistent with a C type nociceptive neuron) and the latter having short action potential durations, no capsaicin sensitivity (consistent with a Axcex4 nociceptive or Axcex1/xcex2 neurons) (Cardenas et al., 1995). There appear to be different types of LVA currents expressed in adult rat sensory neurons based on differential sensitivity to nM concentrations of nimodipine (Formenti et al., 1993). Because of the role of the T type calcium channel in contributing to near threshold membrane excitability, selective suppression of the T channels will decrease neuronal hyperexcitability (painful neuropathies) and raise the threshold for the perception of pain (central pain syndromes).
A specific blocker for T-type calcium channels in the pacemaker cells and conduction fibers in the heart might demonstrate xe2x80x9cpurexe2x80x9d bradycardic (slowing the heart rate) properties since T channels are not usually present in the ventricular myocytes of man. Drugs that block the T-type channel in specific conformational states might allow treatment of tachycardia (by decreasing the heart rate) while having little effect on the inotropic properties of the normal heart (Rousseau et al., 1996). A cardiomyopathic disease (genetic Syrian hamster model) is a result of Ca-overload due to an increased expression of T-type calcium channels in ventricular myocytes (Sen and Smith, 1994). There are increased T-type currents in atrial myocytes from adult rats with growth hormone-secreting tumors (Xu and Best, 1990). A specific T-type calcium channel blocker would act as a cardioprotectant in these cases.
T-type channels in adrenal zona fasciculata cells of the adrenal cortex have been shown to modulate cortisol secretion (Enyeart et al., 1993). Cortisol is the precursor for glucocorticoids and prolonged exposure to glucocorticoids causes breakdown of peripheral tissue protein, increased glucose production by the liver and mobilization of lipid from the fat depots. Furthermore, individuals suffering from anxiety and stress produce too high levels of glucocorticoids and drugs that would regulate these levels are sought after (eg., antagonists to CRF).
T-type calcium channels may be involved in release of nutrients from testis Sertoli cells. T-type calcium channels are expressed on immature rat Sertoli cells (Lalevee et al., 1997). Sertoli cells are testicular cells that are thought to play a major role in sperm production. The intimate juxtaposition of the developing germ cells with the Sertoli cells suggests the latter pay a role in supporting and nurturing the gametes. Sertoli cells secrete a number of proteins including transport proteins, hormones and growth factors, enzymes which regulate germinal cell development and other biological processes related to reproduction (Griswold, 1988). They secrete the peptide hormone inhibin B, an important negative feedback signal to the anterior pituitary. They assist in spermiation (the final detachment of the mature spermatozoa from the Sertoli cell into the lumen) by releasing plasminogen activator which produces proteolytic enzymes. While the role of T channels in not known, they may be important in the release of nutrients, inhibin B, and/or plasminogen activator.
Inhibition of T-type calcium channels in sperm during gamete interaction inhibits zona pellucida-dependent Ca2+ elevations and inhibits acrosome reactions, thus directly linking sperm T-type calcium channels to fertilization (Arnoult et al., 1996).
T-type calcium channels have also been implicated in cellular growth and proliferation, particularly in the cardiovascular system (Katz, 1999; Lijnen and Petrov, 1999; Richard and Nargeot, 1998; Wang et al., 1993).
Tremor can be controlled through the basal ganglia and the thalamus, regions in which T type calcium channels are strongly expressed (Talley et al., 1999). T-type calcium channels have been implicated in the pathophysiology of tremor since the anti-epileptic drug ethosuximide is used for treating tremor, in particular, tremor associated with Parkinson""s disease, essential tremor, or cerebellar disease (U.S. Pat. No. 4,981,867; D. A. Prince).
Pharmacology
There are no known specific blockers of the T-type class of calcium channel. There are ions (ex. Ni+2) that are more effective toward blocking T-type calcium channels vs. HVA channels, and there are a few drugs that block T channels with higher affinity than HVA channels. A number of pharmacological blockers have differential effects on T type calcium currents expressed in different cell types (see Table 1 from (Todorovic and Lingle, 1998)), however there is a diversity of pharmacological profiles of T-type currents. The differential sensitivity of the currents to antagonists may be due to different subunit structure (Perez-Reyes, 1998) as well as cellular environments. T-type calcium channel alpha subunit genes, like the genes for HVA channels, reveal alternative splicing (Lee et al., 1999 Biophys J 76:A408). Extracellular and intracellular loops of individual T-type calcium channel clones show marked diversity amongst themselves and even less homology to HVA channels.
Mibefradil ((1S,2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methyl-amino]ethyl]-6-fluoro-1-isopropyl-1,2,3,4-tetrahydronaphthalen-2-yl methoxyacetate) blocks the T-type calcium channel by preferentially intereacting with inactivated state. Thus, in a cell type with a relatively low RP (xcx9cxe2x88x9250 mV) such as the smooth muscle cells, nearly all T channels will be blocked by mibefradil, whereas in cells with a very negative RP such as cardiac myocytes most of the T channels are not inactivated and therefore will not be blocked by mibefradil (Bezprozvanny and Tsien, 1995). Mibefradil had a complex blocking action on the mouse alpha1G when applied from holding potentials of xe2x88x9260 and could best be fit by fitting to 2 populations of sites (Klugbauer et al., 1999). The high affinity component was reduced at xe2x88x92100 mV. The most prominent (low affinity) site had an IC50 value for mibefradil of xcx9c400 nM.
Ethosuximide is used to treat absence epilepsy and at therapeutically relevant concentrations (0.25-0.75 mM) (Sherwin, 1989) partially blocks T-type currents in some preparations (Coulter et al., 1989). Ethosuximide has different affinities for T-type calcium channels in different tissues. The majority of T type currents from guinea pig or rat ventrobasal thalamic neurons revealed an IC50 for mibefradil of xcx9c500 xcexcM and a maximal block of xcx9c40% block at 1 mM (Coulter et al., 1989). Interestingly, there was no effect of ethosuximide on T-currents in 25% of the TCs tested (Coulter et al., 1989). In hippocampal CA3 neurons, all components of the LVCC were insensitive to ethosuximide at 250 xcexcM or 1 mM. If T-type calcium channels underlie the LVCC in these cells, then the drug had no effect on these T-type calcium channels (Avery and Johnston, 1996). The T-type calcium channels from dorsal root ganglion neurons from one-day-old rats have higher affinity for ethosuximide than thalamic neurons (Kd for T-current is 7 xcexcM vs 15 xcexcM for L-type current) with a maximal block of 100% (Kostyuk et al., 1992). The human alpha1H is insensitive to ethosuximide (Williams et al., WO 9928342; Williams et al., 1999).
Ni2+ is thought to act not only at the pore region but also at another unknown location on the channel protein (Zamponi et al., 1996). The mouse alpha1G has a very low sensitivity to Ni2+ as opposed to other T-type channels (Klugbauer et al., 1999). The human alpha1H expressed in oocytes has an IC50 for Ni2+ of about 6 xcexcM (Williams et al., WO 9928342).
Amiloride, an antagonist at numerous receptors, channels and exchangers, is a low affinity antagonist at T-type calcium channels. There are noted differences in sensitivity of T currents to amiloride (Todorovic and Lingle, 1998). The effects of amiloide are highly variable depending on the cell type, with EC50""s ranging from 50 to  greater than 1000 xcexcM, suggesting that different levels of T-type channel expression in different cells or different channel complexes within different cells (Huguenard, 1996). For instance, the human alpha1H expressed in oocytes has an IC50 for amiloride of about 20 xcexcM (Williams et al., WO 9928342).
NPPB (5-Nitro-2-(3-phenylpropylamino)benzoic acid) has been used to isolate N-type calcium channels (Stea et al., 1999) and was used in studies on the present invention to isolate T-type calcium channels. However, we found NPPB blocked halpha1G-c currents. NPPB has been shown to block voltage-sensitive calcium currents (Kirkup et al., 1996), and, more specifically, L-type calcium currents (Doughty et al., 1998). Interestingly, NPPB reduced the Ca2+ resting current and altered the spike frequency of isolated cockroach dorsal unpaired median neurons (Heine and Wicher, 1998). The resting calcium current may be mediated by a T-type calcium channel, but this has yet to be confirmed.
A DNA molecule encoding a novel isoform of the human T-type low voltage activated calcium channel (alpha1G-c) has been cloned and characterized. The biological and structural properties of this protein is disclosed, as is the amino acid and nucleotide sequence. The recombinant protein is useful to identify modulators of the alpha1G-c calcium channel. Modulators identified in the assays disclosed herein are useful as therapeutic agents and are candidates for the treatment disorders that are mediated by human alpha1G-c activity. Such activities that may be mediated by human alpha1G-c include, epilepsy, schizophrenia, depression, sleep disorders, stress, endocrine disorders, respiratory disorder, peripheral muscle disorders, muscle excitability, Cushing""s disease, fertilization, contraception, disorders involving neuronal firing regulation, respiratory disorders, hypertension, cardiac rhythm, potentiation of synaptic signals, improving arterial compliance in systolic hypertension, vascular tone such as by decreasing vascular swelling, cellular growth (protein synthesis, cell differentiation, and proliferation), cardiac hypertrophy, cardiac fibrosis, atherosclerosis, cardiovascular disorders, including but not limited to: myocardial infarct, cardiac arrhythmia, heart failure and angina pectoris. The recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules.